Collaborating with Dr. Childs of NHLBI we have attempted to monitor allogeneic T cells in patients undergoing allotransplantation. It is crucial to these studies to be able to distinguish between alloresponses and background autoreactivity. To establish T cells are alloimmune, it is essential to be able to compare T cell responses against matched preparations of donor and recipient stimulator leukocytes. Peripheral blood mononuclear cells (MNC) are commonly used for this purpose. Given normal variations and the differences in health status, however, pretransplant peripheral blood MNC from donor and recipient frequently differ markedly in viability and cellular composition. This significantly complicates interpretation, particularly in studying weak T cell responses. To overcome this obstacle, we have begun using cultured donor and recipient B cells as a more uniform, homogeneous source of stimulators. B cells for this purpose can be prepared in high number and purity from both donors and recipients using IL-4 and CD40L, and the resulting cells readily stimulated alloimmune T cell responses. Unexpectedly, using ELISPOT assays, we found the resulting cultured B cell stimulators markedly skew T cell responses towards a type 2 pattern of cytokine response. This could be circumvented using recombinant IL-12. A manuscript describing our finding has been submitted for publication. These observations have important implications, not only for us in developing methods to detect allo-sensitized T cells, but also for investigators using B cells as a convenient and abundant source of APC in a variety of immunologic applications. Pursuing our underlying goals, we are currently using ELISPOT assays to identify T cells directed against alloantigens as described above, and also to detect T cells directed against whole tumor cells, and peptide targets from tumor-associated antigens such as WT1, and PRAME. Of particular interest, in recent studies of MNC from a patient who had experienced dramatic tumor regression of renal cell carcinoma after allotransplanation, we can detect a very vigorous T cell responses against autologous T cells using short term ELISPOT assays. The time course and specificity of this response is under study.